The long term objective of this application is to understand the molecular mechanisms by which allosteric ligands modify enzymatic behavior. Specifically, the allosteric properties of porcine heart fumarase, E. coli carbamoyl phosphate synthetase, bovine heart NAD-dependent isocitrate dehydrogenase, and rat liver phosphofructokinase will be investigated. Fumarase and carbamoyl phosphate synthetase are each regulated by allosteric modifiers that influence maximal velocity and/or Michaelis constants by altering the tertiary structure of the protein. The kinetic behavior of these enzymes will be studied using a thermodynamic linked-function approach. This approach provides for the quantitation of the dissociation constant of modifying ligand from free enzyme, the fractional influence that the modifying ligand has on maximal velocity, and the free energy of interaction between the substrate and allosteric ligand. These latter two parameters describe the efficacy of the allosteric ligand once bound. By observing the changes in these parameters produced by systematically altering the structure of the allosteric ligand or protein, we will be able to infer structural features of the ligand and protein important for these two separate facets of allosteric ligand action. This approach will be extended to investigate the mechanism of action of allosteric ligands that alter an enzyme's aggregation state as well and will be applied to a study of the regulation of isocitrate dehydrogenase by ADP, and phosphofructokinase by a wide variety of regulatory ligands. Isocitrate dehydrogenase should provide a straightforward example of the relationship between tertiary and quaternary levels of allosteric action, whereas phosphofructokinase represents a complex example of several different interdependent regulatory mechanisms. Despite this complexity, significant new insight should be obtained by quantitatively evaluating the pertinent free energy coupling parameters. This insight has been obscured in the past by the common assumption that the enzyme can exist in one of only two possible conformational states. This new insight is especially important in view of the vital role phosphofructokinase plays in carbohydrate homeostasis. The experimental methodologies required for these studies consist primarily of enzyme kinetics and fluorescence polarization measurements, the latter being performed on enzymes covalently labeled with a suitable extrinsic fluorescent probe.